Molecular Characterization of Eimeria Species Naturally Infecting Egyptian Baldi Chickens
Background: Coccidiosis is a serious protozoal disease of poultry. The iden-tification of Eimeria species has important implications for diagnosis and con-trol as well as for epidemiology. The molecular characterization of Eimeria spe-cies infecting Egyptian baladi chickens was investigated.
Methods:Eimeria species oocysts were harvested from intestines of naturally infected Egyptian baldi chickens. The morphometry characterization of oocysts along with COCCIMORPH software was done. The DNA was extracted ini-tially by freezing and thawing then the prepared samples was subjected to commercial DNA kits. The DNA products were analyzed through conven-tional polymerase chain reaction by using amplified region (SCAR) marker.
Results:The PCR results confirmed the presence of 7 Eimeria species in the examined fecal samples of Egyptian baldi breed with their specific ampilicon sizes being E. acervulina (811bp), E. brunette (626bp), E. tenella (539bp), E. max-ima (272bp), E. necatrix (200bp), E. mitis (327bp) and E. praecopx (354bp). A sequencing of the two most predominant species of Eimeria was done, on E. tenella and E. máxima. Analysis of the obtained sequences revealed high identi-ties 99% between Egyptian isolates and the reference one. Similarly, E. maxima isolated from Egyptian baldi chickens showed 98% nucleotide identities with the reference strain. Only single nucleotide substitution was observed among the Egyptian E. tenella isolates (A181G) when compared to the reference one. The Egyptian isolates acquired 4 unique mutations (A68T, C164T, G190A and C227G) in compared with the reference sequence.
Conclusion:This is the first time to identify the 7 species of Eimeria from Egyptian baladi chickens.
Shirley MW, Smith AL, Tomley FM. The biol-ogy of avian Eimeria with an emphasis on their control by vaccination. Adv Parasitol. 2005; 60, 285-330.
Morris GM, Gasser RB. Biotechnological ad-vances in the diagnosis of avian coccidiosis and the analysis of genetic variation in Eimeria. Bio-technol Adv. 2006; 24, 590-603.
McDougald LR. Protozoal infections. In: Saif YM, Barnes, H.J., Glisson, J.R., Fadly, A.M., McDougald, L.R., Swayne, D.E. (Eds.), Dis-eases of Poultry. Iowa State Press, Ames, IA, USA. 2003: 974-985.
FAO. Mapping traditional poultry hatcheries in Egypt. Prepared by M. Ali Abd-Elhakim, Olaf Thieme, Karin Schwabenbauer and Zahra S. Ahmed. AHBL - Promoting strategies for pre-vention and control of HPAI 2009, Rome.
McDougald LR, Fuller L, Mattiello R. A Survey of Coccidia on 43 Poultry Farms in Argentina. Avian The first sentence should be a short brief of what you did and what you got. 1997; 41(4), 923-929.
Andrews RH, Chilton NB. Multilocus enzyme electrophoresis: a valuable technique for providing answers to problems in parasite sys-tematics. Int J Parasitol. 1999; 29, 213-53.
Gasser RB. PCR-based technology in veteri-nary parasitology. Vet Parasitol. 1999; 84, 229-58.
Tsuji N, Kawazu S, Ohta M, Kamio T, Isobe T, Shimura K, Fujisaki K. Discrimination of eight chicken Eimeria species using the two-step pol-ymerase chain reaction. J Parasitol. 1997; 83, 966-970.
Fernandez S, Pagotto AH, Furtado MM, Ka-tsuyama AM, Madeira AM, Gruber A. A multi-plex PCR assay for the simultaneous detection and discrimination of the seven Eimeria species that infect domestic fowl. Parasito. Res. 2003; 127, 317–325.
Reid WM, Long PL. A diagnostic chart for nine species of fowl coccidia. Georgia Uni. Re. Rep. 1979; 335.
Carvalho FS, Wenceslau AA, Teixeira M, Car-neiro JAM, Melo ADB, Albuquerque GR. Di-agnosis of Eimeria species using traditional and molecular methods in field studies. Ve. Para-sitol. 2011b; 176, 95-100. doi: 10.1016/j.vetpar.2010.11.015. Epub 2010 Nov 19.
Casta˜nón C, Fraga J, Fernandez S, Gruber A, Costa L. Biological shape characterization for automatic image recognition and diagnosis of protozoan parasites of the genus Eimeria. Pat-tern Recognitio. 2007; 40, 1899-1910.
Schnitzler BE, Thebo PL, Tomley FM, Uggala A, Shirley MW. PCR identification of chicken
Eimeria: a simplified read-out. Avian Pathol. 1999; 28, 89-93.
Jenkins MC, Miska K, Klopp S. Application of polymerase chain reaction based on ITS1 rDNA to speciate Eimeria. Avian Dis. 2006; 50, 110-114.
Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. J Mol Biol. 1990; 15, 403-410.
Ahmed NE, NegmEldin MM, El Akabawy LM, El Medawy RS. Incidences of some proto-zoan parasites in Birds. KafrElsheikh Vet Med J. 2003; 1 (1), 235-251.
Al-Gawad AA, Mahdy OA, El-Massry AA, Al-Aziz MSA. Studies on Coccidia of Egyptian Balady Breed Chickens. Li Sci J. 2012; 9 (3), 568-576.
Kutkat MA, Shalaby HA, EL.Khateeb RM, Abu ELezz NM, Zayed AA, Abdel Razik AB, Nasef SA, Amer MM. Molecular diagnosis of Eimeria and Clostridia in simultaneously infected chickens. Global Vet. 2009; 3 (1), 26-31.
Lee HA, Hong S, Chunge Y, Kim O. Sensitive and specific identification by polymerase chain reaction of Eimeriatenella and Eimeria maxima, important protozoan pathogens in laboratory avian facilities. Lab Anim Res. 2011; 27 (3), 255-258.
Jatau ID, Sulaiman NH, Musa IW, Lawal AI, Okubanjo OO, Isah I, Magaji Y. Prevalence of Coccidia Infection and Preponderance Eimeria Species in Free Range Indigenous and Inten-sively Managed Exotic Chickens during Hot-wet Season, in Zaria, Nigeria Asian J Poult Sc. 2012; 6(3), 79-88.
Nikam SV, Kanse VS, Jadhav BN, Jaid EL. Comparative study of seasonal incidence of chicken coccidiosis in eight districts of Marathwada region, Maharashtra state, India. Trends in Parasitol Res. 2012; 1 (1), 7-9.
Guven E, Beckstead RB, Kar S, Vatansever Z, Karaer Z. Molecular identification of Eimeria species of broiler chickens in Turkey. Ankara Üniv. Vet Fak Derg. 2013; 60, 245-250.
Long PL, Joyner LP. Problems in identification of species of Eimeria. J Protozool. 1984;31, 535–541.
Ryley JF. Cytochemistry, physiology, and bio-chemistry. In: Hammond, D.M. and Long, P.L. (Eds.), The Coccidia. University Park Press, Baltimore, MD, USA. 1973; 151–154.
Stucki U, Braun R, Roditi I. Eimeria tenella: characterization of a 5S ribosomal RNA repeat unit and its use as a species-specific probe. Exp Parasitol. 1993; 76, 68-75.
Jinneman KC, Wetherington JH, Hill WE, Adams AM, Johnson JM, Tenge BJ, Dang, NL, Manger RL, Wekell MM. Template preparation for PCR and RFLP of amplification products for the detection and identification of Cyclospora sp.And Eimeria spp. oocysts directly from rasp-berries. J Food Prot. 1998; 61, 1497-1503.
Zhao X, Duszynski DW, Loker ES. A simple method of DNA extraction for Eimeria species. J Microbiol Methods. 2001; 44, 131-137.
Abrahamsen MS, Clark TG, White MW. An improved method for isolating RNA from coc-cidian oocysts. J Parasitol. 1995; 81, 107-109.
Tsuji N, Ohta M, Kawazu S, Kamio T, Isobe T, Shimura K, Fujisaki K. DNA polymorphism of srRNA gene among Eimeria tenella strains isolated in Japan. J Vet Med Sci. 1999; 61, 1331-1333.
Sun XM, Pang W, Jia T, Yan WC, He G, Hao LL, Bentue M, Suo X. Prevalence of Eimeria
Species in Broilers with Subclinical Signs from Fifty Farms. Avian Dis. 2009; 53(2), 301-305.
Carvalho FS, Wenceslau AA, Teixeira M, Albu-querque GR. Molecular diagnosis of Eimeria species affecting naturally infected Gallus gallus. Genet Mol Res. 2011a; 10 (2), 996-1005. doi: 10.4238/vol10-2gmr1043.
Morris GM; Woods WG, Richards DG, Gas-ser RB. The application of a polymerase chain reaction (PCR)-based capillary electrophoretic technique provides detailed insights into Eime-ria populations in intensive poultry establish-ments. Mol Cell Probes. 2007; 21: 288-294.
Aarthi S, Raj GD, Raman M, Gomathi-nayagam S, Kumanan K. Molecular prevalence and preponderance of Eimeria spp. among chickens in Tamil Nadu, India. Parasitol Res. 2010; 107(4), 1013-1017. doi: 10.1007/s00436-010-1971-2. Epub 2010 Jul 6.
Meireles MV, Roberto LO, Riera RF. Identifi-cation of Eimeria mitis and Eimeria praecox in Broiler Feces Using Polymerase Chain Re-action. Brazilian J Poult Sci. 2004; 6 (4), 249 -252.
|Issue||Vol 10 No 1 (2015)|
|Chickens DNA Eimeria species Nucleotide PCR Sequencing|
|Rights and permissions|
|This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.|